Microbial contamination and infection is one of the greatest challenges to the survival and well-being of humans and animals, and as such continues to consume huge societal resources. An essential component of the effort to combat pathogens is assessing the presence and viability of prokaryotic and eukaryotic cells. Most commercially available methods capable of assessing cell viability routinely rely on cell growth to make this determination. However, a continuing limitation of these conventional approaches is their dependence on the doubling time of the cell population as well as the practical availability of the proper culturing conditions. Especially in the case of slow-growing cells, methods based on biological growth in order to detect observable changes can require significant time. Therefore, existing methods can be ineffective for applications wherein time delays translate into economic costs and, in extreme cases, human lives. Even where existing detection methods are sufficient, more rapid detection could provide increased efficiencies and reduced costs.
Increasing antibiotic resistance of pathogens has led to a global public health problem manifested in untreatable infections in the human population generally, and on farms. Although the problem is complex, it is known that the increasing use of antibiotics has created selective evolutionary pressures wherein many species of bacteria and infectious protozoa have developed resistance mechanisms, making the antibiotics often prescribed to treat disease no longer effective, and resulting in the spreading of bacterial strains resistant to antibiotics.
A majority of existing methods for determining the antibiotic susceptibility of a pathogen also rely on detection of growth, and rely exclusively on biomass increase due to continuous cell division of the pathogen in culture. Standard plating methods can require many days or even weeks, in the case of slow-growing pathogen, to yield drug-susceptibility results. Optical detection methods, while less time-consuming, still require significant time for the cells to grow to a detectable level. Time delays in obtaining susceptibility test results have led to the clinical practice of empirically prescribing therapies to treat life-threatening infections. The inability to identify antimicrobial-resistant cells in a timely manner results in the prescription of inappropriate therapies and consequently, unfavorable patient outcomes. The continuing emergence of drug resistant strains threatens our ability to treat life-threatening infections through a growing use of ineffective drugs.
There is an ongoing need to reduce the indiscriminate and non-essential use of antibiotics in order to significantly improve patient outcomes and also reduce the spread of bacteria resistant to antibiotics. The disclosed invention provides a means for identifying antibiotic-resistant pathogens rapidly, thereby reducing the number of unnecessarily-prescribed antibiotics.
The ability to detect the presence of harmful cells rapidly and reliably is important for the safe use of numerous medical and industrial products, and the safe and efficient implementation of medical procedures and industrial processes. Rapid determination of water quality during emergency situations, such as floods and earthquakes, immediate diagnosis of trauma patients, screening of raw materials/process equipment in the food industry, monitoring of quality during the pharmaceutical manufacturing phases, and monitoring of biologics and fermentation processes are only a few examples of the applications for the disclosed invention.
Another on-going problem relates to the fact that a functional shelf-life of a unit of platelets, a blood product transfused to control bleeding, is only five days. In order to preserve their physiological function, the platelets must be stored at room temperature. Such conditions are favorable for the growth of many contaminant species of bacteria in the stored units. If not detected, this growth could lead to post-transfusion infection and septic reactions. Methods currently used to establish the sterility of these products require 48 hours time for fast-growing cells, and significantly longer for slow-growing cells in order to grow the cells to detectable levels. Consequently, the effective lifespan of a unit of platelets is reduced to only three days. A more rapid method for identifying fast- and slow-growing contaminant bacteria growing in platelets would increase the useful lifespan of the platelets and place less pressure on an already precious resource.
Bacterial meningitis is an infection causing inflammation of the meninges. In order to recognize bacterial cases wherein a delay in beginning treatment can be life-threatening, effective and rapid diagnosis is essential. Failure to diagnose and treat bacterial meningitis early can result in morbidity with serious, long-term complications including brain damage, hearing loss, learning disability, and death. When a patient presents symptoms of an infection, the physician may prescribe an antibiotic for a suspected bacterial infection before any testing has begun. There is an on-going clinical problem related to the fact that currently available methods cannot effectively culture bacteria in cerebrospinal fluid obtained from “pre-treated” patients, thus making it difficult to confirm the bacterial diagnosis using growth-detecting methods. Rapid and reliable detection of the pathogen and determination of the susceptibility of the pathogen to a particular pharmaceutical agent is of the utmost importance.
One method developed and used to detect the presence of viable cells faster is the impedance sensing of biological samples that measures metabolic deviations to monitor the proliferation of cells and subsequent population growth. Historically, impedance sensing has been used as an electronic analog of the Petri dish to monitor the proliferation of cells and subsequent population growth. Commercially available systems using this approach typically measure either conductance, capacitance, or the full impedance vector, that is, both resistive and reactive components, and use geometries with detection thresholds that require growth up to a million Colony Forming Units per milliliter (106 CFU/ml) or greater in the case of bacteria. However, obtaining that high titer requires significant time, especially if the bacteria belong to a slow-growing species.
Thus, a need exists for new, rapid, and improved methods of detecting viable cells and determining their susceptibility to an external agent or environmental factor. The present invention fulfills this need. Various embodiments of the disclosed invention provide such methods and diagnostic tools that yield test results significantly faster than conventional methods relying on growth or biomass increase.